Since the development of recombinant DNA technology some twenty-five years ago, the prospect of producing proteins on a large scale, rather than extracting them from tissue where they are naturally expressed, has become a reality. In particular, over the last two decades, progress in the development of expression vectors has led to the production of thousands of recombinant proteins on a laboratory scale. Production of commercial quantities of recombinant proteins requires often difficult and expensive scaling up procedures, but has nonetheless also been successful. In addition, transgenic animals including mice, rabbits, pigs, sheep, goats and cows have been engineered to produce human pharmaceuticals in their tissues or secretions. Houdebine, L. M., J. Biotechnology 34:269-287 (1994).
Although egg white is thought to be an excellent host for recombinant protein production, preparing transgenic avians has proven to be technically difficult due in large part to problems involved in manipulating the chicken embryo. When oviposition occurs, the embryo has already reached a stage corresponding to a mammalian late blastula or early gastrula. Genetic manipulation of the embryo during earlier development requires reintroduction to the female or in vitro culture, both difficult procedures. Houdebine, L. M., J. Biotechnology 34:269-287 (1994). Despite these difficulties, transgenic chickens have been produced that are resistant to infection by avian leukosis (Crittenden and Salter, “Transgenic Livestock Models In Medicine And Agriculture” pp. 73-87 (Wiley-Liss (1990))), or have high levels of circulating growth hormone. Bosselman, R. A., et al., Science 243:533-535 (1989).
Four general methods for generating transgenic avians have been reported. One method involves excision of a developing egg from the oviduct, microinjection of DNA near the blastoderm, and in vitro culture of the manipulated embryo in solution and surrogate shells. Love, J., et al., Biotechnology 12:60-63 (1994). A second method requires the culture and transfection of primordial germ cells, with subsequent transplantation into an irradiated recipient near the same stage of development as the donor. Carsience et al., Development 117:669-675 (1993); Etches et al., Poultry Science 72:882-889 (1993). Although technically very demanding, these two approaches are attractive because large pieces of DNA can be transferred.
A third method involves blind injection of replication competent retrovirus with a needle near the blastoderm of a newly laid egg. Petropoulos, C. J., et al., J. Virol. 65:3728-3737 (1991). Although this method is the simplest, it is also limited in that the DNA to be transferred must be approximately 2 kb or less in size and, the method results in viremic hens which shed infective recombinant retrovirus. Petropoulos, C. J., et al., J. Virol. 66(6):3391-3397 (1992).
The fourth method involves a replication-defective retroviral vector system (see, e.g., U.S. Pat. Nos. 5,162,215 and 4,650,764, hereby incorporated by reference). One of these systems (Watanabe and Temin, Mol. Cell. Biol. 3(12):2241-2249 (1983)) has been derived from the reticuloendotheliosis virus type A (REV-A). Sevoian et al., Avian Dis. 8:336-347 (1964). Replication-defective retroviral vectors derived from the REV-A virus are based on the helper cell line C3 (Watanabe and Temin, Mol. Cell. Biol. 3(12):2241-2249 (1983)) which contains the components of a packaging defective helper provirus. The derivation of the C3 helping line and several replication-defective retroviral vectors have been described in detail in U.S. Pat. No. 4,650,764 and Watanabe and Temin, Mol. Cell Biol. 3(12):2241-2249 (1983). This method is more technically demanding than the replication competent technique in that the blastoderm must be exposed, and microinjection equipment must be used. Bosselman, R. A., et al., Science 243:533-535 (1989). Nonetheless, it results in transgenic hens free of replication competent retrovirus, and can transfer DNA as large as 8 kb in size.
Tissue specific expression of a foreign gene in a transgenic chicken was achieved using the replication competent retrovirus technique. Petropoulos, C. J., et al., J. Virol. 66(6):3391-3397 (1992). A replication competent retrovirus was used to deliver the reporter gene chloramphenicol acetyl transferase (CAT), driven by a muscle specific promoter, a action, to skeletal muscle. Tissue specific expression of a recombinant protein in the egg of a transgenic avian has not yet been successful.
It would thus be desirable to provide a vehicle and method for transferring a gene to an embryonic chicken cell (or other avian species) so as to create a transgenic hen wherein the gene is expressed in a tissue specific manner. It would also be desirable to provide a vehicle and method for transferring a gene to an embryonic chicken cell, wherein the gene is expressed in the hen's oviduct and secretion of the gene product is in the hen's eggs. It would also be desirable to provide a vehicle and method for transferring a gene to an embryonic chicken cell, wherein the gene is expressed in the hen's oviduct and secretion of the gene product is in the hen's eggs without compromise to the hen's health and the health of other birds in contact with her.